1. Field of the Invention
The present invention relates to an apparatus for determining the base sequences of nucleic acids according to the Sanger method by labeling primers with a pigment such as a fluorescent substance or phosphorescent substance first and spectroscopically reading the sequence from fragments as electrophoresed on a gel in the final step utilizing the luminescence from the labeling pigment.
2. Description of the Prior Art
The bands of nucleic acid fragments as developed by gel electrophoresis can be read utilizing fluorescence by two systems, i.e. on-line system and off-line system.
With the on-line system, nucleic acid fragments are electrophoresed on a gel, and during the electrophoresis, variations with time in the intensity of fluorescence of a point on the lane are read. With the off-line system, a gel of electrophoresed fragments is mounted after electrophoresis on a specific reading device to read the electrophoretic pattern.
According to the Sanger method (see Proc. Natl. Acad. Sci. U.S.A., vol. 74, p. 5463(1977)), four kinds of nucleic acid fragments wherein the terminal base is A (adenine), G (guanine), T (thymine) or C (cytosine) are used as a set of samples. When it is attempted to electrophorese on one lane one kind of sample having one of the four kinds of terminal bases, or to electrophorese many samples at the same time, the off-line system must measure the fluorescence of a slab of electrophoretic gel in two-dimensional directions, while even the on-line system requires one-dimensional high-speed fluorescence measurement in the direction of arrangement of the samples on the electrophoretic gel.
The known fluorescence measuring devices for slabs of electrophoretic gel are all of the on-line type.
Fluorescence measurement can be realized most simply using the apparatus shown in FIG. 9. (The apparatus described in "High Technology," December 1986, page 49 also belongs to this category.)
With reference to FIG. 9, a polyacrylamide gel 2 is immersed at its opposite ends in an electrolyte in electrode tanks 4 and 6. A voltage is applied across the electrode tanks 4, 6 from a power supply 8. One end of the gel 2 is formed with slots 10 for the injection of samples. Samples of different terminal bases are injected into the slots 10. The voltage applied from the power supply 8 electrophoreses the samples through the gel 2 in the direction of arrow 12 for development.
A laser 14 serving as an excitation light source emits an exciting beam, which is reflected at a half mirror or dichroic mirror 16 and projected on the gel 2 through an objective lens 18. The fluorescence from the fluorescent label on the sample migrating through the gel 2 is collected by the objective lens 18 again, transmitted through the half mirror or dichroic mirror 16 and then through a fluorescence selecting interference filter 20, impinges on a photomultiplier tube 22 serving as a photoelectric device and is thereby detected.
With the apparatus of FIG. 9, the single objective lens 18 is used both for projecting the exciting beam and for receiving the fluorescence, and the gel 2 is mechanically scanned with the overall optical system including the objective lens 18 and the components associated therewith in the direction 23 of arrangement of the samples (i.e. in a transverse direction perpendicular to the direction 12 of electrophoresis in the illustrated case).
FIG. 10 shows another apparatus for measuring the fluorescence of a slab of electrophoretic gel 2 (see the Proceeding of 24th Annual Meeting of the Japanese Biophysical Society in Japan, 3E 1130, October, 1986).
The exciting beam from a laser 14 serving as an excitation light source is made to incident by a condenser lens 24 on an end face of the gel 2 in a direction parallel to the gel. The fluorescence is received through a lens 26 one-dimensionally or two-dimensionally at once in a direction normal to the plane of the gel 2, passed through a fluorescence selecting interference filter 20, amplified by an image intensifier 28 and made to incident on a one- or two-dimensional photosensor (array type sensor) 30 for detection.
The apparatus of FIG. 9 for determining base sequences is adapted to measure the fluorescence in the direction of reflection of the exciting beam, so that Rayleigh scattering of the exciting beam provides intense background light to result in an impaired S-N ratio. Rayleigh scattering occurs intensely toward the front and rear but diminishes in a direction at an angle of 90 degrees with the exciting beam.
Further with the apparatus of FIG. 9, the objective lens 18, as well as the excitation optical system and the light-receiving optical system must be mechanically moved wholly or partly for scanning. For on-line measurement, it is required that all the lanes be scanned within a period of time which is sufficiently short relative to the speed of electrophoresis, whereas such a precision optical system is generally heavy, great in inertia and in no way adapted for high-speed scanning. Even if so adapted, the system will then be very costly.
In the case of the apparatus of FIG. 10, the electrophoretic gel 2 is exceedingly great relative to the diameter of the fluorescence receiving lens 26 which is usually usable, with the result that the solid angle of the fluorescence received is extremely small, giving a feeble fluorescence detection signal, which must be compensated for by using a one- or two-dimensional sensor and amplifying the output greatly. For this purpose, there arises a need to use, for example, the image intensifier 28, which nevertheless is very expensive.
Further if the gel 2 is thin, it is likely that the laser beam will not be confined in the gel. Another problem is also encountered in that unless the gel is accurately planar, the exciting beam will be bent upon incidence thereon, failing to afford any measurement.